The inactivation of gene functions by reverse genetic material is the most important methods for switching off certain genes. This is of great importance for combating infectious and other diseases (including AIDS), caused by interference with gene expression. A gene function can be inactivated on various levels: by homologous recombination at the DNA level, by antisense nucleic acids or ribozymes on the RNA level, or by antibodies on the protein level. In conversion to practice, all four possibilities have advantages and disadvantages. For therapeutic applications, only the RNA inactivation by antisense molecules, or by ribozymes appears to be implementable. Both classes of compounds can be synthesized chemically or produced in conjunction with a promotor by biological expression in vitro or even in vivo. The principle of catalytic self-cleavage of RNA molecules and the cleavage in trans has become well established in the last 10 years. The hammerhead ribozymes are characterized best among the RNA molecules with ribozyme activity. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it seems clear that catalytic antisense sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site.
The basic principle of constructing ribozymes is very simple. An interesting region of the RNA, which contains the GUC (or CUC) triplet, is selected. Two oligonucleotides strands, each with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them.
Molecules of this type were synthesized for numerous target sequences. They showed catalytic activity in vitro and in some cases also in vivo. The best results were obtained with short ribozymes and target sequences. A topical challenge for the in vivo application is the construction of ribozyme genes which permit a continuous expression of the ribozyme in a particular cell (Bertrand, E. et al., (1994) Nucleic Acids Res. 22, 293 to 300).
There are five possible reasons for interference with a satisfactory functioning of expressed ribozymes within the complex intracellular milieu.
1. The mRNA substrate exists within the cell presumably in a highly folded structure, which furthermore can also be protected by proteins bound to parts of the structure. The encountering of accessible sites within the substrate allowing for hybridization with the complementary flanking regions of the ribozyme is a question of actual probability. Computer-aided predictions of possible, thermodynamically stable secondary structures can be useful when searching for loop regions without base pairing. However, the physiological relevance of these conformation models is still uncertain.
2. Since the target mRNA is transported immediately out of the cell nucleus, the ribozyme must also enter the cytoplasm, preferably along the same path. It is, however, difficult to achieve a co-localization of ribozyme and its substrate.
3. The in vivo use of ribozymes requires the insertion of ribozyme genes in suitable expression cassettes. The transcription of these constructs can produce mRNAs, in which the central, catalytic, secondary structure of the ribozymes is displaced by other, more stable base pairings within the non-complementary flanking sequences.
4. 100- to 1,000-fold excess of ribozyme molecules relative to the target sequence is necessary for attaining a recordable increase in the RNA level. The production of 10.sup.5 to 10.sup.6 ribozymes per cell over a long period of time can, however, have cytotoxic effects. In general, such high expression levels are not stable. An excess of ribozymes is needed because of the inadequate stability of the ribozymes in the presence of nucleases, because of the ineffective transport to the cytoplasm and because of the less than optimum conversion factor of the cleavage reaction.
5. The kinetics of the cleavage reaction and the ability of the ribozymes to carry out multiple conversion reactions depends on the binding parameters and the structure of the complementary flanking regions of the ribozymes. Cellular proteins can affect the catalysis of the cleavage reaction, probably with the help of the dissociation of the ribozyme from the substrate of the cleavage reaction, which represents the preliminary step of the next cleavage reaction. Until now, it has not been possible to predict the optimum structure of the flanking regions for a ribozyme, to guarantee high specificity and high conversion. Despite many efforts to construct specific ribozyme genes, generally only partial successes have been achieved, mostly on the basis of trial and error experimentation.